CD44 is a Type I transmembrane glycoprotein expressed by virtually every cell in the vertebrate body. CD44 is a cell surface adhesion molecule, which has been shown to be involved in various processes, such as in cell trafficking, cell migration, cell homing, cell-cell interactions and cell-matrix interactions. The N-terminus of CD44 includes the extracellular ligand-binding domain of the molecule. Various ligands are known to interact with CD44. Hyaluronic acid (HA) is the principal ligand of CD44 (Miyake et al., J Exp Med 1990, 172:69-75, Aruffo et. al., Cell 1990, 61: 1303-13, Peach et. al., J Cell Biol 1993, 122:257-64), however, additional extracellular matrix (ECM) components (such as laminin, collagen, fibronectin and chondroitin sulfate (Jalkanen et. al., J Cell Biol 1992, 116:817-25, Faassen et. al., J Cell Biol 1992, 116:521-31) as well as non-ECM constituents (mucosal vascular addressin, serglycin, osteopontin and class II invariant chain), E- and L-selectin (Dimitroff et. al., PNAS 2000, 97:13841-6 and Dimitroff et. al., J Cell Biol 2001, 153:1277-86) and aggrecan (Fujimoto et. al., Int Immunol 2001, 13:359-66) may also interact with the CD44 receptor. Marked accumulation of CD44, and sometimes hyaluronic acid, is detected in areas of intensive cell migration and cell proliferation, such as in wound healing, tissue remodeling, inflammation (including auto inflammation), morphogenesis and carcinogenesis. The juxtamembrane portion of the cytoplasmic tail of CD44 binds to members of the ezrin-radixin-moesin (ERM) family of actin linker molecules, thus providing a connection between cell surface bound CD44 and the actin cytoskeleton (Tsukita et. al., J Cell Biol 1994, 126: 391-401), thus establishing the basis for CD44-dependent cellular motility.
Depending on the species, the CD44 locus contains about 20 coding exons. For example, the Human CD44 gene includes a total of 19 exons, while the mouse CD44 gene includes 20 exons. The exons of CD44 may be classified into two classes: constant exons and variable exons. The constant exons of both human and mouse include exons C1-C5 at the 5′ terminus and C6-C9 at the 3′ terminus and encode for the so-called constant regions of CD44 (Screaton et. al., PNAS 1992, 89: 12160-4; Tölg et. al., Neucleic Acids Res, 1993 21:1225-9; Screaton et. al., J Biol Chem 1993, 268: 12235-8). The variable exons are located in the middle of the molecule and include 9 exons in humans (exons V2-V10) and 10 exons in mice (exons V1-V10). The variable exons encode for the variable regions of CD44. The main molecular species expressed in cells is the standard, hematopoietic, form of CD44 (also named CD44s or CD44H), which is the shortest form of CD44 and is encoded by a mRNA (messenger ribonucleic acid) consisting exclusively of constant exons. Retention of different combinations of variable exons in the mRNA results in a myriad of CD44 splice variants (CD44v, reviewed in Gunthert, Curr. Top Microbiol Immunol 1993, 184:47-63). However, although in theory more than 1,000 individual splice variants may be produced this way, and even in cases where multiple splice variants are co-expressed in one tissue or cell type, CD44s remains the main isoform (for example, Ni et. al., J. Lab. Clin. Med 2002, 139: 59-65; Bell et. al. MCB 1998, 18:5930-41). Under various circumstances and conditions, splicing patterns of CD44 are often altered (such as for example in Gunthert et. al., Cell 1991, 65:13-24; Heider et. al., J Cell Biol 1993, 120:227-33; Wielenga et. al., Cancer Res 1993, 53:4754-6). CD44 transcripts may be produced which contain additional exons; for example, unskipped exons V3, V5, V6 and V7 are known to be expressed by activated lymphocytes and metastatic variants of tumor cells (Naor et al, Crit Rev Clin Lab Sci, 2002, 39:527-79). The cell's choice of the CD44 splice variant is the main determinant for the binding affinity (Lesley et. al., J Exp Med 1995 182:431-7, Stamenkovic et. al. Embo J 1991 10: 343-8, Van der Voort et al., Biochem Biophys Res Commun 1995 214: 135-144).
Expression of various CD44 splice variants in several disease conditions, such as autoimmune diseases, was studied and has been proposed as a target for both diagnoses and treatment in such diseases. Monoclonal antibodies (mAbs) directed against various variant regions of CD44 were suggested as potential agents for treatment of autoimmune diseases. Reber et. al. describe mAbs directed against metastasis-specific variants of CD44V surface protein of a rat pancreatic adenocarcinoma (Reber et. al. Int J Cancer, 1990, 46:919-27). Anti-CD44 monoclonal antibodies, which inhibit T-cell proliferation, were also provided for treatment of various autoimmune diseases (Rothman et. al. J Immunol. 1991 147:2493-9). Monoclonal antibodies specific for variant forms of CD44 containing exon v6 were also reported as being useful for diagnosing lymphoma (Ristamaki et. al. Blood, 1994, 84:238-43). In addition, it has been reported (Haynes et. al. Arthritis Rheum, 1991, 34:1434-43) that administration of a CD44 protein, peptide or derivative can be used for treating various autoimmune diseases. CD44 expression is also a known target for anti-tumor and anti-inflammatory therapies. Experiments in animals have shown that targeting of CD44 by antibodies, antisense oligos and CD44-soluble proteins markedly reduce the malignant activities of various neoplasms. Antisense strategies and various oligonucleotide-based therapies directed against CD44 expression have been developed, such as described for example in U.S. Pat. No. 6,150,162 and U.S. Pat. No. 5,990,299.
There is some evidence that CD44 and some of its splice variants are involved in autoimmune and pathogen induced neurological disorders, such as, for example, Multiple Sclerosis (MS) and Human T-cell lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Studies of a mice strain with Experimental Allergic Encephalomyelitis (EAE), frequently used as an animal model of Multiple Sclerosis, show that CD44 is induced in vivo on glial cells surrounding inflammatory lesions. (Haegel et. al., J Cell Biol. 1993, 122:1067-77). Mononuclear cells which express the CD44V10 splice variant were detected in the spinal cord of EAE mice (Laman et. al., Mult Scler. 1998, 4:147-53). Animals vaccinated with CD44V3-V10 cDNA developed significantly less severe EAE when compared with sham vaccinated animals or animals vaccinated with CD44s cDNA. (Garin et. al., J Neurol Sci. 2007, 258:17-26). In vivo treatment with an antibody against CD44s did not affect the disease burden whereas combined treatment with antibodies against the isoforms containing the variable regions 6, 7 and 10 (CD44V6, V7 and V10), reduced the disease burden considerably (Laman et. al., Mult. Scler. 1998, 4:147-53).
Human T-cell lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is caused by HTLV-I infection and characterized by spastic paraparesis and urinary disturbance with perivascular HTLV-I-infected and activated CD4+ T-cell infiltration. A CD44 splice variant which contain a direct connection between exons V6 and V10 (CD44V6/V10) was found to be frequently expressed in peripheral blood mononuclear cells of patients of HAM/TSP, (Matsuoka et. al., J Neuroimmunol. 2000, 102:1-7) These findings led to speculation that the V6/V10-containing lymphocytes are able to migrate into the CNS with ease even at early stages of the disease (Matsuoka et. al., J Neuroimmunol. 2000, 102:1-7).
The “classic” neurodegenerative disorders (such as Alzheimer's disease (AD), Parkinson's Diseases (PD) and Amyotrophic Lateral Sclerosis (ALS)) are adult onset, chronic, progressive and irreversible severely disabling diseases. Additional, non-autoimmune neurodegenerative disorders may include, for example, motor neuron disorders (MND), such as, for example, but not limited to: primary lateral sclerosis (PLS) and Spinal Muscular Atrophy (SMA). Alzheimer's disease (AD) is characterized by progressive mental and cognitive deterioration with consequent formation of amyloid plaques, neurofibrillary tangles, gliosis and neuronal loss. The disease occurs in both genetic and sporadic forms whose clinical course and pathological features are quite similar. Three genes have been identified to date which, when mutated, cause an autosomal dominant form of Alzheimer's disease. These genes encode the Amyloid Protein Precursor (APP) and two structurally and functionally related proteins, presenilin-1 (PS1) and presenilin-2 (PS2). Mutations in any of the three proteins enhance proteolytic processing of APP via an intracellular pathway that produces Amyloid β peptide (Aβ), a 40-42 amino acid long peptide that is the primary component of amyloid plaque in Alzheimer's disease.
Amyotrophic Lateral Sclerosis (ALS) is a progressive lethal neurological disease affecting one to two in every 100,000 people who are diagnosed with ALS each year. ALS occurs when the motor nerve cells that control voluntary movement gradually degenerate. The loss of these motor neurons causes the muscles which they control to weaken and waste away, leading to paralysis and eventually death. The etiology of most ALS cases remains unknown, but 2% of instances are due to mutations in the Cu/Zn superoxide dismutase gene (SOD1). In the latter, mutant SOD1 induces non-cell-autonomous motor neuron killing by an unknown gain of toxicity. Selective vulnerability of motor neurons likely arises from a combination of several mechanisms, including protein misfolding, mitochondrial dysfunction, oxidative damage, defective axonal transport, excitotoxicity, insufficient growth factor signaling, and inflammation. Damage within motor neurons is enhanced by damage incurred by non-neuronal neighboring cells, via an inflammatory response that accelerates disease progression (Boillee et. al. Neuron. 2006, 52:39-59, Pehar et. al., Neurodegener. Dis. 2005, 2:139-46).
Parkinson's disease (PD) is a chronic and progressive neurodegenerative disease caused by a selective degeneration of dopaminergic neurons in the substantia nigra pars compacta of the brain; 80% of the neurons die of an unknown cause before the symptoms appear. Symptoms include intermittent tremor in the limbs, poor balance and difficulty in initiating movement.
Primary Lateral Sclerosis (PLS) is a rare neuromuscular disease characterized by progressive muscle weakness in the voluntary muscles. As a motor neuron disease, PLS usually develops when nerve cells, which control voluntary muscle movement, degenerate and die, causing weakness in the muscles they control. Spinal Muscular Atrophy (SMA) is a term applied to various disorders, all having in common a genetic cause and the manifestation of weakness due to loss of the motor neurons of the spinal cord and brainstem.
Unlike neurological autoimmune diseases (such as multiple sclerosis), in which the innate immune system targets normal neuronal cell constituents through infiltration of T-cells and lymphocytes across the blood brain barrier, in classic neurodegenerative disorders the neurons die of an unknown reason. The role of the neuron-glia interaction and the inflammatory process in classic neurodegenerative diseases has been suggested. Macro and microglial cells have been suggested in having a role in multistep degenerative processes in ALS and respective disease models. The activation of astroglial and microglial cells occurs early in the pathogenesis of the disease and seems to greatly influence disease onset and promotion (Di Giorgio et. al., Nat Neurosci. 2007; 10:608-614; Esposito et. al. Exp Neurol. 2007; Kim et. al., Exp Mol Med. 2006; 38:333-47).
No clear evidence of the role of specific variants of CD44 in Alzheimer disease, Amyotrophic Lateral Sclerosis or Parkinson's disease has been provided. In primary cultures of mouse astrocytes, surface expression and mRNA levels of CD44 could be induced via stimulation with either phorbol ester (PMA), or tumor necrosis factor alpha plus gamma interferon. The CD44 transcripts produced contain additional exons, including the exon v6, as well as variants of larger size. However it is not known if such activation occurs in vivo or in humans in the course of neurodegenerative diseases (Haegel et. al, J Cell Biol, 1993, 122:1067-77). The localization of CD44s was investigated by immunohistochemistry in postmortem human brain tissue of control subjects and patients with Alzheimer's disease. In gray matter, it was found to be associated with some astrocytes of both protoplasmic and fibrous morphology. In the Alzheimer's disease brain, the number of CD44 positive astrocytes increased dramatically. CD44 may be an important adhesion molecule for these astrocytic processes (Akiyama et. al., Brain Res. 1993, 632:249-59). However, it is not known if cells expressing CD44 participate in the disease progress or are helping to prevent neurodegeneration. No CD44 splice variants were described in these diseases.